5'Hoxd genes play many roles during limb development and may control the effectors of morphogenesis at late stages. How Hoxd genes guide digit morphogenesis and their downstream targets remain enigmatic. We find that, in addition to a role in initiating sonic hedgehog expression, 5'Hoxd genes determine the posterior polarity of the primary limb axis early, and regulate digit pattern and morphogenesis at late stages, after digit condensations have already formed, including joint formation and positioning; a major mechanism by which Hoxd genes regulate digit identity. We have also discovered genetic and physical interactions between 5Hoxd and Gli3 that modify Gli3R function (and hence Shh output), antagonizing Gli3R and potentially converting it to an activator. Gli3-Hox interactions may modulate Gli3 repressor activity and activate targets in other Shh-dependent contexts, such as normal or neoplastic renewal of skin and gut epithelia. Gli3-Hox interactions also play a role in regulation of cartilage vs joint formation, which may have relevance for the homeostasis of the skeletal system and skeletal diseases, as well as skeletal birth defects. What is the cellular role of Gli3-Hoxd interaction? Hoxd transcription factors cooperate in an additive fashion to regulate digit pattern and are thought to be key targets of Shh signals. We previously found that Hoxd-Gli3 interactions serve to modify the function of Gli3 as a nuclear Sonic hedgehog-mediator either by converting Gli3-repressor into an activator of its target promoters and/or antagonizing Gli3 repressor function. Collaborating with Chris Westlake (CCR,NCI), we are extending this finding by examining Hox-Gli3 interaction in vivo with fluorescent imaging to assess whether interaction plays a role only in the nucleus, or at other sites where Gli proteins are functionally processed in response to hedgehog signaling (specifically cilia). While Hoxd genes are no longer expressed in the adult, other related Hox genes are expressed, have highly conserved in Gli3-binding domains and may modify Hh-Gli3 targets in other contexts, such as skin and gut, during normal renewal of these epithelia or during neoplastic proliferation. We have also determined residue requirements for Gli3-HoxD protein interaction and are testing the functional effects of a dominant interfering form of Gli3 (peptide) in chick embryos. What role does Hoxd-Gli3 interaction play in determining polarity of the primary limb axis? In most vertebrates, the primary limb axis runs through the posterior limb with the ulna/digit4 (d4) condensing first. In urodele amphibians such as axolotl, which retain the ability to regenerate limbs as adults, the anterior limb axis is dominant (radius/d2 appear first). Based on altered expression patterns, it has been proposed that the axis shift in Urodeles results from a failure to expand 5'Hoxd gene expression in the late distal limb. We have analyzed limb axis formation in the 5'Hoxd mutant (Hoxd11-13 deleted) and found that the anterior axis forms first as in urodeles. Furthermore, we find that in compound 5'Hoxd;Gli3 mutants, posterior axial dominance is restored. The 5'Hoxd homeobox transcription factors play roles in replication licensing and cell adhesion. Gli3 repressor, expressed anteriorly, also regulates proliferation and condensation, and antagonizes 5'Hoxd function. We are currently analyzing how changes in the relative timing and rate of proliferation and of cell aggregation/condensation in different zones of the limb bud are altered in these mutants, and if they correlate with anterior vs posterior axial dominance. We propose that the balance between antagonistic 5'Hoxd-Gli3 functions governs the polarity of primary limb axis formation and are investigating the potential relation between altered axis polarity and regenerative capacity. What role do Hoxd genes play in cartilage differentiation and joint formation? Digit identity remains plastic even after theformation of the digit primordial chondrogenic condensations and is regulated by interdigit zones, which are also late sites of 5'Hoxd and Gli3 expression. We found that genetic removal of several Hoxd genes (d11-d13) results in abnormal joint formation, both loss of digit joints and/or abnormal positioning of joints, as well as brachydactyly (short, biphalangeal digits). The canonical Wnt signaling pathway is known to play an essential role in joint formation and we find that activated beta-catenin restores normal joint formation in the 5'Hoxd mutant digits. But surprisingly, selective activation of stabilized beta-catenin in the interdigital tissues is required for rescue, suggesting that at least some aspects of beta-catenin and 5'Hoxd function in joint formation occur indirectly, via interdigit signaling. Gli3 (the transcriptional effector of sonic hedgehog, and interactor with Hoxd proteins) also has striking effects on cartilage differentiation and joint formation in digits. During joint formation in digit precursors, Gli3 mutants form abnormal segments with excessive, abnormal joint formation extending into the cartilage elements. Reducing the 5'Hoxd dosage by half (deleting one genomic locus) completely rescues this phenotype, allowing formation of normal joints and digits with the normal 3 bony segments. Our genetic evidence indicates that the Hoxd-Gli3 balance acts indirectly, from interdigital mesenchyme, to modulate Bmp pathway activity and thereby regulate the periodic appearance of digit elements (phalanges) and joints from a digit tip progenitor pool. We plan to extend our analysis to determine: 1) target promoters regulated by Gli3-Hoxd interaction and 2) other signaling inputs that regulate the digit tip progenitor pool to determine phalanx number and size. What signaling pathways interact with Hoxd genes to regulate final digit morphogenesis? Digit shape and numbers of joints are regulated at late stages by interdigit signals. Our genetic evidence indicates that 5'Hoxd and Gli3 genes are part of an interdigit signaling center that regulates final digit identity. Elucidating signaling pathway differences between different interdigits will provide new insights on how digit identity is regulated at late stages and the potential mechanisms by which Hoxd and Gli3 genes act. We are evaluating interdigits in species with evolutionary digit adaptations, to correlate morphogenetic changes with changes in signaling activity, comparing three vertebrates: chick, mouse, and bat (collaborators J. Rasweiler, SUNY; Marian Ros, Univ. Cantabria). Both bats and birds have evolved striking digit adaptations for flight and also have highly adapted hindlimbs including changes in phalanx number and joint formation. We are undertaking a global analysis of gene expression using RNAseq to screen for differences in various signaling pathways between individual interdigit samples at the RNA expression level (data analysis collaborative effort with Dr. Agarwala, NCBI). Comparing gene expression in the interdigits and responsive digit condensations of different organisms with very different digit morphologies will provide new insights on how digit identity is regulated and evolutionary adaptation occurs. Global expression profiling analyses will also be applied to 5'Hoxd mutants and following rescue (joint formation restored by beta-catenin activity or Gli3 loss) to gain further insight into critical signaling pathways regulating digit morphology and implicated in cartilage growth and joint segmentation. These findings will also be highly relevant to the problems of congenital malformations and skeletal regeneration.